Aspartic acid is one of the amino acids used by animals in the synthesis of proteins, and as such only L-aspartic acid is utilized. Although it is not an essential amino acid, which is to say that animals can synthesize this amino acid, it is nonetheless used as a feed additive, especially in Japan where production of synthetic L-aspartic acid in 1978 was 500-1,000 tons. Impacts of Applied Genetics: Micro-Organisms, Plants and Animals, Office of Technology Assessment. Aspartic acid also is used in seasoning industries. But whatever growth in L-aspartic acid production could be expected to result from the aforementioned uses is dwarfed by its anticipated growth as a component in the dipeptide sweetener, L-aspartyl-L-phenylalanine methyl ester. The demand of L-aspartic acid for this use alone is estimated at 1,000 metric tons per year by 1985 and double that by 1990.
A traditional chemical synthesis of L-aspartic acid has been frustrated by the necessity of resolving a racemic mixture, which is too costly to permit a commercially feasible process. Alternately, the obstacle to a total chemical synthesis of L-aspartic acid may be viewed as arising from the absence of a chiral catalyst which selectively synthesizes the L-enantiomer, or which would selectively destroy the D-enantiomer in a racemic mixture. However, nature has provided chiral catalysts in the form of enzymes, and enzymatic methods are the bases of L-aspartic acid production.
Aspartase is an enzyme which catalyzes the conversion of fumaric acid and ammonia to L-aspartic acid, as well as the reverse reaction of deamination of L-aspartic acid, that is, ##STR1## Aspartase itself can be produced by many micro-organisms, although not necessarily in quantities usable for a commercial process. Thus, U.S. Pat. No. 3,214,345 describes a method of producing L-aspartic acid by fermentation in a medium containing fumaric acid and anmonia using micro-organisms including Pseudomonas fluorescens, Pseudomonas aeroginosa, Bacillus subtilis, Bacillus megatherium, Proteus vulgaris, Escherichia coli and Aerobacter aerogenes. The patentees state that an important and critical feature of their invention is that the fermentation medium be sugar-free. The reason for this limitation is that whereas sugars promote the growth of the micro-organisms they concurrently repress aspartase production. A non-fermentative process for aspartic acid synthesis is decribed in U.S. Pat. No. 3,791,926 where the patentee immobilized an aspartase-producing micro-organism in a matrix formed by polymerizing a water-soluble monomer in an aqueous suspension of microbial cells. In comparing the synthetic methods based on immobilized cells with those based on immobilized aspartase the patentees found that substantial loss of enzyme activity occurred in extraction of aspartase from cells and subsequent concentration of the enzyme from the crude extract. The patentees thus developed and preferred a method of synthesis based on immobilized whole cells to obviate serious disadvantages encountered using immobilized aspartase itself.
In conceptualizing a method for producing L-aspartic acid several criteria can be elaborated. One is that the enzyme should be produced in high yield by a micro-organism which grows quickly using inexpensive growth media under non-stringent biological conditions. Another criterion is that the process should be heterogeneous so as to maximize utilization of aspartase. Because immobilized whole cells impose transport limitations arising both from the matrix itself and from the transport of substrate and product across the cell membrane, a method based on an immobilized enzyme is preferable. However, the latter method has additional requirements including the facile rupture of cell walls to release aspartase, stability of aspartase outside of the cell, and relative ease of purification, or at least concentration, of aspartase without catastrophic loss of enzyme or enzyme activity.
As is discussed more fully herein, I have succeeded in obtaining a micro-organism which grows rapidly under ordinary biological conditions using relatively inexpensive nutrients and which expresses aspartase, in part constitutively, in relatively high yields. The aspartase thus produced can be readily isolated and purified with good recovery, and is immobilized with relatively high efficiency. The resulting immobilized aspartase gives excellent production of L-aspartic acid from fumaric acid and ammonia over its usable lifetime.
An important advantage of the enzyme-producing microorganism of this invention is that maximum enzyme production and microbial growth can be obtained within about 7-8 hours, compared to a period of about 3 days when using a suitable Escherichia coli. Another important advantage, both unexpected and surprising, is that the micro-organism of this invention is quite sugar tolerant, which is to say that aspartase formation is not repressed by sugars even at levels of about 1.0 percent, and in fact aspartase production is increased by sugars such as glucose at levels up to about 1.0 percent. The advantage which accrues from such sugar tolerance is that aspartase can be formed via microbial growth using a relatively cheap and widely available energy source, namely, sugars. Still another advantage is the aspartase produced by the micro-organism of this invention is at least partly constitutive. Thus, although it is desirable to grow the micro-organism in the presence of, for example, L-aspartic acid to maximize enzyme production, L-aspartic acid is not an indispensable requirement for cellular aspartase production.